Detection or concentration determination of various hormones, proteins, medicines and metabolites thereof involved in body fluids or excreted fluid has played an important role as clinical analysis in diagnosis of diseases, judgement of prognosis and decision of therapeutics. Such determination or quantitative analysis has been made physicochemically, biochemically or immunologically. Above all immunological methods such as radioimmunoassay (RIA), enzyme immunoassay (EIA) have widely been used in the clinical analysis since a very small ammount of substances contained in body fluids or urine may be determined specifically and in good reproducibility. Method for detection of a very small amount of substances in body fluids or urine according to immunological agglutination or agglutination inhibition reaction has also widely been used.
The method by virtue of agglutination or agglutination inhibition is carried out for instance by injecting an antigen into a mammal to produce the antibody specific thereto, applying the antigen and/or antibody to fine carrier particle such as red blood cells, latex polymer particles to be adsorbed, reacting with antigen or antibody, and macroscopically observing or instrumentally determining the state of agglutination or agglutination inhibition so as to recognize presence or absence of antigen or determine concentration thereof.
The so-called "nonspecific agglutination reaction", which means agglutinations other than agglutination specifically caused by immunological antigen-antibody reaction and which is caused by some substances existing in body fluids or urine, may considerably influence on the method of detection according to the agglutination or agglutination inhibition reaction using such carrier. This comes into question particularly in case of the method wherein antibody adsorbed by carrier is reacted with antigen in test sample so as to detect presence or absence of agglutination of the sensitized carrier or quantitatively analyze concentration of antigen. When such nonspecific agglutination occurs, there is a possibility that false judgement holding existence of the antigen in body fluid or urine despite of absence thereof or lower concentration thereof despite of higher concentration of antigen is given, which may considerably influence on the precision and the reliability of the method as to detect presence or absence of antigen, or determine concentration of antigen and consequently on diagnosis of diseases, judgement of prognosis or decision of therapeutics.
It is thus very important by the reasons referred to above to exclude the nonspecific agglutination reaction in the method for detecting antigen in body fluid or urine by means of immunological agglutination or agglutination inhibition reaction. Hithertofore various methods have been proposed for excluding such undesired reaction, among which are method specifying pH value and kinds of buffer solution for the reagent (Japanese Early Opened Patent Application Gazettes Nos. 35754/82 and 182168/82), method filtering to remove the substance causing such nonspecific agglutination reaction (Japanese Early Opened Patent Application Gazettes Nos. 31696/72, 146022/80, 182170/82; Japanese Patent Publication Gazettes Nos. 43038/77, 24509/82), method adding specific additive to the reagent to be used (Japanese Early Opened Patent Application Gazettes Nos. 82230/75, 12419/80, 1970/82, 9723/82, 59167/82; Japanese Patent Publication Gazettes Nos. 12741/68, 11407/74) and method decomposing antibody with using hydrolytic enzyme (Japanese Early Opened Patent Application Gazette No. 139595/79).
Although many proposals have been made for excluding undesired nonspecific agglutination which is very interested in the field of clinical analysis, any of said methods are not satisfactory in that it is difficult to completely and selectively exclude such undesired reactions, that specific agglutinative ability is lost even if the reaction can selectively be excluded that antibody structure which is recognized as a cause of nonspecific agglutination with substances such as rheumatoid factor and complement, or that very complicated preparation of the reagent is necessitated for attaining the purpose.
On the otherhand, chemical modification of protein with an acylating agent has widely been carried out for the purpose of study of structure or improvement of thermal stability of protein (Japanese Patent Publication Gazette No. 29039/82). As for the other methods for chemical modification of antibody, substitution with 2, 4-dinitrophenyl group (Japanese Early Opened Patent Application Gazette No. 139595/79) and succinylation ("Nature" Vol. 210, page 536, 1966) has been known. The former is aimed at strong adsorption of antibody to fine carrier particle and the latter relates to preparation of succinylated .gamma.-globulin and fundamental study of nature thereof. At any rate it has never been known yet that acylated antibody is effective for excluding immunologically nonspecific agglutination or agglutination inhibition reaction.
The inventors have tried to develop a reagent for readily and quickly detecting antigen contained in body fluids or urine according to immunological agglutination reaction without causing undesired nonspecific agglutination to find out that it is possible to astonishingly completely exclude influence by substances in body fluids or urine to cause the nonspecific agglutination by using fine carrier particle sensitized with acylated antibody so as to make it possible to detect antigen in test sample in high precision, on which finding the present invention is based.